Enzymatic fluorometric assay for cAMP and adenylate cyclase

ABSTRACT

The present invention relates to a method for quickly determining CAMP content or an adenylate cyclase activity in a biological sample containing non-cyclic adenine nucleotides without the use of radioactive agents. Particularly, the present invention provides a method of determining CAMP content or an adenylate cyclase activity in a biological sample containing non-cyclic adenine nucleotides selected from the group consisting of CAMP produced by endogenous adenylate cyclase, and AMP, ATP, ADP and a mixture thereof, which comprises (1) combining a biological sample with effective amounts of apyrase, adenosine deaminase and alkaline phosphatase to enzymatically remove non-cyclic adenine nucleotides other than CAMP, and glucose-6-phosphate in the sample; (2) enzymatically converting CAMP into AMP; (3) determining an amount of AMP without the use of radioactive agents, and a kit to carry out the method.

TECHNICAL FIELD

The present invention provides a method of determining cAMP content oran adenylate cyclase activity in a biological sample containingnon-cyclic adenine nucleotides selected from the group consisting ofCAMP produced from ATP by endogenous adenylate cyclase (cyclicadenosine-3′,5′-monophosphate), ATP (adenosine-triphosphate), ADP(adenosine-diphosphate) and a mixture thereof without the use ofradioactive agents. More particularly, the invention relates to a methodwhich comprises: (1) combining a biological sample with effectiveamounts of apyrase, adenosine deaminase and alkaline-phosphatase toenzymatically remove non-cyclic adenine nucleotides other than cAMP, andglucose-6-phosphate in the sample; (2) enzymatically converting cAMPinto AMP; (3) determining an amount of AMP without the use ofradioactive agents.

Adenylate cyclase (adenylyl cyclase, adenylate cyclase, EC4.6.1.1) is anenzyme catalyzing the conversion:

ATP→cAMP in the presence of Mg²⁺ or Mn²⁺.

Adenylate cyclase exists locally on cell membranes and plays a criticalrole as a signal transduction cascade of a number of fundamentalhormones and neurotransmitters.

For example, measurement of adenylate cyclase activity has been employedto study the altered physiology exhibited by transplanted human heartsand in congestive heart failure. See M. R. Bristow et al., New Engl. J.Med., 307, 205 (1982); K. G. Lurie et al., J. Thorac. Cardiovasc. Surg.,86, 195 (1983).

Adenylate cyclase activity can be determined by monitoring the changesof cAMP content synthesized from ATP by the catalytic action ofadenylate cyclase.

However, a more clear elucidation of the biological role of adenylatecyclase has been limited by the difficulty in monitoring accuratelychanges in the tissue level of cAMP.

cAMP (cyclic adenosine-3′,5′-monophosphate) was found as a factor whichintermediates blood sugar rising action of adrenaline and glucagon inliver cells. [E. W. Sutherland et al., J. Am. Chem. Soc., 79, 3608(1957)]. And also CAMP was found to intermediate actions of hormonessuch as adrenocorticotropin (ACTH), luteinizing hormone (LH), tyrosinestimulating hormone (TSH) and parathyroid hormone (PTH) or physiologicalactive substances such as prostaglandin. Thus, when peptide hormones oractive amines have been secreted and have reached at target cells, cAMPtransfers Information for them to proceed enzymatic reactions, that is,plays a role of a second messenger.

cAMP is synthesized from ATP by adenylate cyclase located on membranesin the living body and decomposed by phosphodiesterase into 5′-AMP. cAMPis present widely in bacteria or animals but the concentration of cAMPis extremely low (a stationary concentration is 0.1-1 nmol/g wetweight). As an assay of CAMP, an assay using CAMP binding protein orradioimmunoassay is conveniently employed. The CAMP content depends oneutrophy, proliferation, differentiation, adaptation of cells andchanges in sensitivity.

Measurement of cAMP in a wide variety of mammalian and non-mammaliantissue and fluids provides a useful way to assess cell viability,endocrine hormonal axis function, adenylate cyclase activity andphosphodiesterase activity. In addtion, measurement of cAMP can be usedto evaluate the activity of a number of signal transproduction proteins,including, but not limited to, the family of G proteins(guanine-nucleotide binding protein) which play a major role in signaltransduction, ribosomal protein synthesis, translocation of nascentproteins and other important cellular functions. Bourne et al., Nature,348, 125 (1990).

Furthermore, measurement of cAMP may be used in evaluating otherendogenous and exogenous compounds (for example, nitrous oxide) whichmay alter the level of cyclic nucleotides in a particular cell, tissue,organ or body fluid.

Many hormones use cAMP as a second messenger including, but not limitedto: epinephrine, norepinephrine, adrenocorticotropin (ACTH),vasopression, glucagon, thyroxine, and thyroid-stimulating andmelanocyte-stimulating hormones which are some of the principleregulatory hormones/proteins in the living organism. The activity of allof these hormones and regulators can be measured in tissues, serum, bodyfluids, and in all cell cultures (cells and medium) using the method forcAMP of the present invention. Measurement of these hormones isperformed in a wide variety of disease states where hormonal imbalancemay lead to specific pathology.

Once a hormone or regulatory protein interacts with a specific receptor,the second messenger, in this case, cAMP, is produced through a cascadeof biochemical events. The production of cAMP can also be specificallyinhibited in some cases by hormones which use a decrease in cAMP as partof the specific hormonal signal-transduction pathway. The result of thisregulatory protein or hormone and receptor interaction can be, but isnot limited to, (1) an alteration in cell permeability secondary, forexample, to changes in ion channels, (2) and alteration in the rate ofenzyme catalyzed reactions sensitive to he concentration of cAMP, and(3) an alteration in the rate of protein synthesis including thesynthesis and degradation of other enzymes. a content of cAMP can beused to directly and indirectly monitor the consequences afterinteracting a hormone or regulatory protein with a receptor.

Specifically, cAMP can be measured in urine or blood for use as a markerfor drug levels, like aminophylline or theophylline which stimulate theadrenergic nervous system by preventing the breaKdown of endogenouscAMP. Measurement of cAMP in cell cultures can be used to assessspecific hormones, regulatory protein and drugs where cAMP represents avital link in the signal transduction process.

cAMP can also be used to assess cell viability and stability by studyingcells in the absence or presence of a specific hormone or regulatoryprotein. For example, measurement of cAMP in liver cells (hepatocyte) byglucagon, can be used to assess hepatocyte viability. This may beuseful, for example, in organ and/or cell transplantation, for exampleheart, liver, lung, kidney, pancreas, skin and brain celltransplantation.

Measurement of the responsiveness of cells from biopsy samples afteractivation by a wide variety of hormones, regulatory proteins and drugwhich either increase or decrease cellular cAMP levels, can be used as away to specifically assess cell function.

A specific clinical example is the use of cAMP measurement in cardiacbiopsies to assess the responsiveness of myocardium. Cardiomyopathicheart cells do not respond with the same rise in cAMP content afterβ-adrenergic stimulation as normal heart cells. The diagnosis of theseverity of the heart disease and the efficacy of some drugs, such asβ-adrenergic blockers and angiotensin converting enzyme Inhibitors, canbe made comparing the responsiveness of biopsy samples from normalhearts to cardlomyopathic hearts. Measurement of basal and/or stimulatedlevels of adenylate cyclase activity or cAMP in blood cells can be usedto guide therapy in such patients. In addition, release of cAMP eitherintracellarly or into the arterial or venous circulation can be used asan indicator of the response of an organ and/or tissue to a variety ofdifferent physiologic and nonphysiologic stresses such as ischemia,hypoxia, or drug or hormonal stimulation. Tissue or body fluid levels ofcAMP can be measured in nearly ever mammalian cell or body fluid,including blood cells and platelets, with this approach. In sometissues, cAMP levels can be measured in response to specific stimulatorsas an index on oncogenicity and/or invasiveness, in the case of samplesof potentially tumorous cells. In other cases, measurement of cAMP canbe used to determine the effectiveness of specific therapies which mayalter cAMP synthesis or degradation.

As described above, cAMP plays an important role as a second messengerin information transfer in cells as well as has also variousphysiological functions. It is significant in fields of basic andclinical medicine to measure cAMP synthesized from ATP by a catalyticaction of adenylate cyclase in order to determine activity of adenylatecyclase or elucidate behavior of cAMP.

BACKGROUND ART

Measurement of adenylate cyclase activity is carried out by quantitativedetermination of cAMP produced from ATP as a substrate. Methods formeasurement of cAMP are grouped into two methods using as a substrate(1) labeled ATP and (2) non-labeled ATP.

In the method using labeled ATP as a substrate (1), using ATP labeled bya radioactive element, for example, [α-³²P] ATP, as a substrate, andcAMP ([³²p] cAMP) generated from radioactively labeled ATP is separatedand determined.

See Y. Salomon et al., Anal. Biochem., 58, 541 (1974; R. A.

Johnson et al., In Method in Enzymology, 195, 3 (1991)).

The method employs sequential affinity chromatography with ionicexchange resin and aluminum oxide columns for separation of [³²p] cAMPfrom [α-32P] ATP.

Although this method is sensitive, it relies upon dangerously and costlyradioactively labeled compounds.

On the other hands, the methods using non-labeled ATP are classifiedinto (1) radioimmunoassay wherein radioactively labeled cAMP issubjected to antigen-antibody reaction competitively with anti-serumincluding cAMP generated from non-labeled ATP and then radioactivity ofbinding antibody is assayed to determine cAMP content, and (2)protein-binding assay wherein radioactivity of ³H-cAMP bound withcAMP-dependent protein kinds is measured using specific binding betweencAMP-dependent protein. kinase and cAMP. See A. G. Gilman et al., Proc.Natl. Acad. Sci. USA, 67, 305 (1970).

The method using the substrate non-labeled ATP can not compensatedecomposition of cAMP by cyclic nucleotide phosphodiesterase andtherefore the method is not appropriate for samples including strongphosphodiesterase activity.

Given the safety and environmental concerns, the use of radioactivematerials should be avoided. A need exists for a highly sensitivenon-radioactive assay to measure adenylate cyclase activity and cAMP asan index of adenylate cyclase activity.

However, it is very difficult to determine cAMP without usingradioactive compounds because of the extremely low concentration of cAMPin most mammalian issues. In addition, since non-cyclic adeninenucleotides such as cAMP, ADP and ATP in a biological sample are presentin several hundred to several hundred thousand times the concentrationof cAMP and also those chemical structures are similar to the that ofcAMP, they act as interfering substances in assay of cAMP. Particularly,ATP is present in one hundred million times the concentration of cAMPand therefore it is substantially impossible to exactly determine cAMPwithout complete removal of endogenous ATP.

On the other hand, cAMP is converted into AMP by action ofphosphodiesterase. An assay for AMP without radioactive compounds hasbeen disclosed. Lowry et al. have developed a sensitive assay based onthe fluorescence of reduced pyridine nucleotide. See O. H. Lowry et al.,A Flexible System of Enzymatic Analysis, Harcourt Brace Jovanovich, NewYork (1972); F. M. Matschinsky et al., J. Histochem. Cytochem., 16, 29(1968). The assay depends on it that absorbency of reduced nicotinamideadenine dinucleotide phosphate (NADPH) at 340 nm is 0.617 per 0.1 mmoland an absolute concentration of NADPH is calculated on absorbency of asample.

An assay for AMP is disclosed which depends upon the stimulatory effectsof AMP on glycogen phosphorylase, the enzyme that converts glycogen intoglucose-1-phosphate in the presence of inorganic phosphate (P₁). See E.Helmrich et al., Biochemistry, 52, 647 (1964); ibid., 51, 131 (1964); M.Trus et al., Diabetes, 29, 1 (1980). According to the method, glycogenphosphorylase activity is determined by an amount of glucose-1-phosphategenerated from glycogen and AMP can be assayed using the glycogenphosphorylase activity as an index. Lurie also have developed asensitive assay for AMP. See K. Lurie et al., Am. J. Physiol., 253, H662(1987).

A method to increase the analytical sensitivity and specificity for cAMPor adenylate cyclase have employed enzymic degradatiom of non-cyclicadenine nucleotides or their removal by chromatography. See N. D.Goldberg et al., Anal. Biochem. 28, 523 (1969); B.Mcl. Breckenridge,Proc. Natl. Acad. Sci. USA, 52, 1580 (1964).

In the conventional analysis, since interfering endogenous ADP or ATPcould not be completely removed, it has been considered that measurementof cAMP should be impossible. Therefore, there has been substantially nomethod for highly sensitive measurement of cAMP content and adenylatecyclase activity based on the amount of cAMP without using radioactivesubstances.

From a completely different point of view, the inventor and otherspreviously attempted to develop assays for adenylate cyclase activityand cAMP and as a results of an intensive study, they had found a methodfor highly sensitive measurement of cAMP content and adenylate cyclaseactivity based on the amount of cAMP with using only enzymatic andchemical reactions, which comprises removing selectively interferingsubstances, endogenous non-cyclic adenine nucleotides, such as ATP, ADPand the like using enzymes, converting AMP into ATP, converting ATP intoglucose-6-phosphate through fructose-6-phosphate, converting NADPH,determining NADPH concentration and correlating with cAMP concentration(WO94/17198)

According to the method, cAMP at an amount of μg order in a biologicalsample can be strictly measured at a level of pmol or fmol. See A.Sugiyama et al., Anal. Biochem., 218, 20 (1994); A. Sugiyama et al., J.Clin. Lab., 8, 437 (1994); A. Sugiyama et al., Anal. Biochem., 225, 368(1995); A. Sugiyama et al., Yamanashi Med. J., 10, 11 (1995).

The reaction schemes of the conventional method above are describedbelow.

The method as stated above was very excellent as a method forquantitative analysis in a principle of methodology and theoreticallycorrect. However the method takes long time for a cleaning reaction or areaction mixture gets cloudy when an enzyme has been deactivated byheating after a cycle reaction.

DISCLOSURE OF INVENTION

The present invention has been accomplished as a result of intensivestudy for development of a simple and fast method for determining cAMPand adenylate cyclase without radioactive substances.

The present invention has improved on the methods of determiningadenylate cyclase activity and quantitative analysis for cAMP withenzymatic reactions and fluorescence intensity which the inventor andothers previously developed (WO94/17198). That is, (1) in CleaningReactions by deleting of 5′-nuleotidase from the enzymes to be used, 1hour of the reaction period can be extremely shortened to from 5 to 10minutes; (2) in Cycling Reactions, enzymes used are deactivated byremoving Mg²⁺ with a chelating agent such as EDTA, instead of heating.The reaction mixture does not get cloudy and accuracy of DetectingReaction on the next step is improved; (3) Converting Reaction ischanged to 1 step reaction from conventional 2 steps convertingreactions. The operation can be simpler; and (4) the concentrations ofreaction agents used in Detecting Reactions were reviewed to optimize inthe present method. By these improvements, an enzymatic fluorometricassay or a spectrophotometric assay in which cAMP and adenylate cyclasecorresponding to cAMP are determined quickly and in high sensitivitycould be provided.

Incidentally, as described below, the present method can be used formeasurement of guanine regulatory proteins and cAMP specificphosphodiesterase.

The reactions used in the present method for determining cAMP are shownbelow.

The reactions illustrated in the above schemes are further demonstratedbelow.

Step 1—Cleaning Reactions (Removal of endogenous non-cyclic adeninenucleotides and glucose-6-phosphate)

The present method for measurement comprises steps in which endogenouscompounds having non-cyclic adenine group other than cAMP (adenosine,ATP, ADP and AMP) are enzymatically removed by a mixture of apylase,adenosine deaminase and alkaline phosphatase. Preferably, the presentmethod comprises a step in which glucose-6-phosphate in a sample isenzymatically converted into glucose by using alkaline phosphatase(Cleaning Reactions).

The Cleaning Reactions of the present invention can remove allendogenous ATP, ADP and AMP which are present in much higherconcentrations than cAMP and substantially increase the backgroundsignal. Since glucose-6-phosphate generates during subsequent DetectingReactions, it is favorable to enzymatically remove glucose-6-phosphatefrom a sample previously for improving the precision of measurement. TheCleaning Reactions are important for raising sensitivity.

Also, the Cleaning Reactions period was extremely shortened andsimplified by deleting 5′-nucleotidase from four kinds of enzymes, i.e.apyrase, 5′-nucleotidase, alkaline phosphatase and adenosine deaminasewhich- have been used in a conventional cleaning reaction step. That is,it has been found that about 1 hour of the conventional cleaningreaction period can be shortened to only 5 to 10 minutes.

Step 1—Optional cleaning reactions 1 (Removing of fructose-6-phosphate)

It is favorable to remove fructose-6-phosphate, which is generatedduring Cycling Reactions and Detecting Reactions, from a samplepreviously by hydrolysis with alkaline phosphatase.

Step 1—Optional Cleaning Reactions 2 (Removal of endogenous glycogen ina sample)

It is more favorable to remove from a sample endogenous glycogen, whichis converted to glucose-6-phosphate by using glucose oxidase, glycogenphosphorylase and alkaline phosphatase (Optional reactions). Accordingto this Optional Cleaning Reactions, endogenous glycogen which is aninterfering substance in Detecting Reactions, wherein a known amount ofglycogen is added, is destroyed.

Step 2—Converting reaction (Conversion to AMP)

Subsequently, phosphodiesterase is combined with the reaction mixtureafter the Cleaning Reactions so that cAMP is converted to AMP(Converting Reaction).

Step 3—Detecting Reactions (Fluorometric assay of NADPH)

After the Converting Reactions, glycogen and inorganic phosphoric acidare added to the reaction mixture and an amount of AMP is determined bycorrelating a level of glucose-6-phosphate which is finally generatedfrom glycogen with glycogen phosphorylase activated by AMP.Glucose-1-phosphate is converted to glucose-6-phosphate withphosphoglucomutase and finally alucose-6-phosphate is enzymaticallyconverted into 6-phosphogluconolactone, NADPH and H⁺. The concentrationof NADPH is measured by fluorometric assay , for example, according tothe method of Trus et al. [M. Trus et al., Diabetes, 29, 1 (1980)].

Step 3—Optional Detecting Reactions (Degradation of6-phosphogluconolactone)

And also, 6-phosphogluconolactone can be converted into6-phosphogluconate by heating in an aqueous solution in vitro, and then6-phosphogluconate can be converted into NADPH and ribulose-5-phosphatein the presence of NADP⁺ in vitro. The concentration of NADPH can beincreased by the Optional Reaction and measured by fluorometric assay,for example, according to the method of Trus et al. as described above[M. Trus et al., Diabetes, 29, 1 (1980)].

Where stimulated adenylate cyclase activity was measured in the samepreparation from rabbit heart with both the modified Salomonradioactivity method and the present fluorometric method withoutalkaline phosphatase, the results were similar. Weign et al., Anal.Biochem., 208, 217 (1993). Result with the radioactive assay arecomparable to the present fluorometric method. Although the absolutespecific activities are different when the results from the radioactiveassay and the fluorometric assay are compared, the fold stimulation ofadenylate cyclase as determined using either method is similar. Thedifferences in specific activities are most likely due to minor factorsin adenylate cyclase reaction mixtures. That is, unlabelled cAMP is usedin the radioactive assay to prevent [³²P] cAMP degradation by endogenousphosphodiesterases, whereas theophylline is used in the fluorometricassay to inhibit endogenous phosphodiesterase degradation of newlysynthesized cAMP.

Moreover, measurement of adenylate cyclase using ATP-ADP Cyclingreactions as shown in Step 3—Detective Cycling Reactions 2, revealedthat the absolute specific activities were the same for both theradioactive and fluorometric assays.

The method of the present invention is conveniently carried out by a kitpreviously prepared, and such a kit comprises vials containing enzymes,buffer solutions and the like to be used at each reaction step.

Specifically, a kit for performing a method for quickly determining thecAMP content or the adenylate cyclase activity of the present inventionis exemplified by a kit which comprises (1) a vial for the cleaningreactions comprising effective amounts of apylase, alkaline phosphataseand adenosine deaminase to remove endogenous non-cyclic adeninenucleotides consisting of ATP, ADP and AMP, and endogenousglucose-6-phosphate in a biological sample; (2) a vial for theconverting reaction comprising an effective amount of phosphodiesteraseto enzymatically convert cAMP in the biological sample to AMP; and (3)vials for the detecting reactions comprising effective amounts of (a)glycogen, inorganic phosphoric acid and glycogen phosphorylase toconvert glycogen to glucose-1-phosphate, (b) phosphoglucomutase toconvert glucose-1-phosphate to glucose-6-phosphate, and (c)glucose-6-phosphate dehydrogenase and NADP(β-nicotinamide adeninedinucleotide phosphate)⁺ to convert glucose-6-phosphate to6-phosphogluconolactone and NADPH.

Also, another kit for performing a method for quickly determining thecAMP content or the adenylate cyclase activity of the present inventionmay be a kit which comprises (1) a vial for the cleaning reactionscomprising effective amounts of apylase, adenosine deaminase andalkaline phosphatase to enzymatically remove endogenous non-cyclicadenine nucleotides consisting of ATP, ADP and AMP, and endogenousglucose-6-phosphate in a biological sample; (2) a vial for theconverting reaction comprising effective amounts of phosphodiesterase,ATP, myokinase, phosphoenol pyruvate and pyruvate kinase toenzymatically convert cAMP in the biological sample to AMP; and (3)vials for the detecting reactions comprising effective amounts of (a)fructose and hexokinase to convert ATP to fructose-6-phosphate and (b)phosphoglucose isomerase, glucose-6-phosphate dehydrogenase and NADP+toconvert fructose-6-phosphate to 6-phosphogluconolactone and NADPH.

The method of the present invention is sensitive enough to measure cAMPin small biological samples weighing less than 0.1 mg and can be adaptedto measure 0.1 fmol cAMP/sample. According to the present invention, aknown amount of ATP is added to a biological sample and then the ATP isconverted into cAMP by action of adenylate cyclase in the sample.Adenylate cyclase activity may be provided by determining the convertedAMP after removing all adenine nucleotides such as endogenous ATP byCleaning Reactions.

Ammonium ion produced from adenosine in Cleaning Reaction drives thecleaning reaction sequence essentially to completion, preventingreformation of nucleotides in the sample. These steps provide for asignificant unexpected improvement over fluorometric assays. Weign etal., Anal. Biochem., 208, 217 (1993).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a line graph showing the fluorescence against concentration ofATP in various incubation periods.

FIG. 2 is a graphical plot of the absorbance against concentration ofstandard samples of cAMP as determined by an embodiment of theinvention.

FIG. 3 is a histogram showing the amounts of cAMP detected in untreatedand stimulated cells as determined by the present fluorometric method(solid) and commercially available immunocolorimetric assay (shaded) andradioimmunometric assay (clear).

FIG. 4 is a histogram showing the adenylate cyclase activity determinedby the present fluorometric method (solid) and commercially availableradioimmunometric assay (shaded) and Salomon method (clear).

BEST MODE FOR CARRYING OUT THE INVENTION

The physiological material which is assayed with the present method ispreferably obtained from a mammalian source, including tissue, bloodcell, bone and physiological fluids such as urine, blood, spinal fluidand the like and may be fresh or frozen. Lowry et al., A Flexible Systemof Enzymatic Analysis, Harcourt Brace Jovanovich, New York (1972).

Thus, in a preferred embodiment, the present invention comprises thesteps of:

Step 1—Cleaning Reaction (Removal of endogenous non-cyclic nucleotidesand glucose-6-phosphate)

A sample of a physiological material comprising cAMP,glucose-6-phosphate and at least one non-cyclic adenine nucleotide,i.e., selected from the group consisting of ATP, ADP, AMP and mixturethereof s combined with an aqueous buffer comprising a mixture orapyrase, adenosine deaminase, and alkaline phosphatase so that saidnon-cyclic adenosine nucleotides (the ATP, ADP and/or AMP) areenzymatically converted to remove.

The wording “more quickly” means that the period of Cleaning Reactionwhich has conventionally taken about 1 hour is shorten within about 5 to10 minutes, i.e., by from a twelfth to a sixth, at least a sixth of thereaction period.

Step 1—Optional Cleaning Reactions 2

And optionally, the reaction mixture is combined with a mixture ofglucose oxidase and glycogen phosphorylase and alkaline phosphatase sothat all glycogen in the sample is destroyed, while the cAMP is retainedin the reaction mixture. These steps destroy all endogenous glycogenwhich is an interfering substance in Step 3—Detecting Reaction(Fluorometry of NADPH) in which a known amount of glycogen is added.

Step 2—Converting Reactions (Reaction of conversion to AMP)

The reaction mixture is combined with phoshodiesterase, so that saidcAMP is converted to AMP.

Step 3—Detecting Reactions (Fluorometric assay of NADPH)

Further said AMP is contacted with glycogen phosphorylase in a presenceof glycogen and inorganic phosphatase so that glucose-1-phosphate isproduced in the reaction mixture. Then the glucose-1-phosphate isenzymatically converted into o-phosphogluconolactone, NADPH and H⁺ insaid reaction mixture and the concentration of NADPH is fluorometricallymeasured. The concentration of NADPH is correlated with theconcentration of adenylate cyclase activity, cAMP content or aconcentration of AMP.

The method of determining cAMP is based on a principle that AMP producedby cleavage of 3′,5′-phosphodiester bond of cAMP stimulates glycogenphosphorylase activity. That is, the amount of cAMP and adenylatecyclase activity correspond to the absorbance of NADPH finally obtainedfrom glycogen phosphorylase activity which is activated by AMP producedfrom cAMP.

The correlation of a final concentration of NADPH with a concentrationof AMP can be obtained, for example, by a calibration curve. See FIG. 2.

Preferably, following the Cleaning Reactions, the enzymes used in theCleaning Reaction may be deactivated by heating.

Further preferably, following Step 1—Cleaning Reactions, a solution ofphosphodiesterase, glycogen-phospholylase, glucose-1,6-diphosphate,inorganic phosphate, glycogen, NADP⁺, glucose-6-phosphate dehydrogenase,phosphoglucomutase and Mg²⁺ is added to the reaction mixture and Steps 2of Converting Reactions and Step 3 of Detecting Reactions 1 are carriedout sequentially, in situ.

Step 3—Optional Detecting Reactions 1 (Degradation ofphosphogluconolactone)

The concentration of NADPH which is generated in Step 3 can be increasedby sequentially converting the 6-phosphogluconolactone to6-phosphogluconate by heating the reaction mixture of Step 3—DetectingReactions 1 and then reacting the 6-phosphogluconate with added NADP⁺and 6-phosphogluconate dehydrogenase in the presence of Mg²⁺ to yieldribulose-5-phosphate, NADPH, H⁺ and CO₂.

Step 3—Detective Cycling Reaction 1

The effective concentration of NADPH which is generated in Step3—Detecting Reactions 1 can be increased in orders of magnitude byemploying it in cycling reaction system. One such reaction systemconverts NADPH added to α-ketoglutarate into NADP and glutamate. TheNADPH⁺ in turn converts added glucose-6-phosphate into6-phosphogluconolactione and NADPH. As described above, the6-phosphogluconolactone can be hydrolyzed (H₂O, heat) and converted toribulose-5-Phosphate and NADPH in the presence of 6-phosphogluconatedehydrogenase. Lowry et al., A Flexible System of Enzymatic Analysis,Harcourt Brace Jacovanovich, New York (1972).

Step 2—Optional Converting Reactions 1 (Reaction of Conversion to ATP)

Alternatively, the AMP which is produced from cAMP in Step 2—ConvertingReactions can be converted to ADP by combining it with a trace amount ofATP in the presence of myokinase. The ADP which is produced is thenconverted to ATP and pyruvate by combining the ADP with2-phospho(enol)pyruvate kinase. One molecular AMP produces one molecularATP in this step (Reaction of converion to ATP).

Step 2—Converting Reactions and Step 2—Optional Converting Reactions 1,in which cAMP is converted to AMP, and AMP is converted to ATP throughADP, can be carried out sequentially at one step.

Step 3—Detecting Reactions 2 (Fluorometric assay of NADPH)

Further, ATP is converted to fructose-6-phosphate and ADP by combiningwith hexokinase In the presence of fructose. Fructose-6-phosphate isconverted in the presence of phosphoglucoisomerase toglucose-6-phosphate, which is enzymatically converted to6-phosphogluconolactone, NADPH and H⁺. The concentration of NADPH ismeasured by the method of Trus et al. as described above. M. Trus etal., Diabetes, 29, 1 (1980).

Step 3—Optional Detecting Reactions 1

Alternatively, as described above, the 6-phosphogluconolactone can behydrolyzed and further converted to 6-phosphogluconate, which can beconverted to NADPH, H⁺, CO₂ and ribulose-5-phosphate.

Step 3—Detective Cycling Reactions 2 (Amplification of ATP-ADP cyclingreaction)

Further, ATP is subjected to ATP-ADP cycling reaction. That is, ATP isconverted to fructose-6-phosphate and ADP by combining it withhexokinase in the presence of fructose.

Then the ADP is converted to ATP and pyruvate by combining phosphoenolpyruvate in the presence of pyruvate kinase.

The ATP thus produced is again converted to fructose-6-phosphate andADP. By repeating of these reactions, fructose-6-phosphate can befinally accumulated.

A chelating agent is added in order to deactivate enzymes at the end ofthe Cycling Reactions. The chelating agents can be exemplified bypolyaminocarboxylic acid such as EDTA, oxycarboxylic acid such as citricacid, preferably, EDTA.

The resulting fructose-6-phoshate, produced from the excess fructose andphospho(enol)pyruvate used in the Cycling Reactions, is converted toglucose-6-phosphate using phosphoglucose isomerase, and theglucose-6-phosphate is converted into 6-phosphoaluconolactone and NADPHby exposing the glucose-6-phosphate dehydrogenase and NADP⁺. The NADPHconcentration can then be determined by Trus et al. method. M. Trus etal., Diabetes, 29, 1 (1980). See Step 3—Detecting Reactions 2 asdescribed above.

Step 3—Detecting Reactions 3

According to the present invention, it is possible toabsorptiometrically detect the ATP produced as shown in Step 2—OptionalConverting Reactions 1 with chemiluminescence assay that utilizes thelucifelase reaction and firefly lucifern. Wulff et al., Methods ofEnzymatic Analysis, Bergmeyer H.U., eds., VCH (1985). From ATPdetermination, it is possible to calculate the AMP concentration and thecAMP concentration and adenylate cyclase activity corresponding to them.

Although the rate of this reaction is very slow, the yield of thereaction (defined as the ratio of the number of emitted photons and thenumber of converted ATP molecules) is almost 100%. The intensity of theemitted light is directly proportional to the ATP concentration and ismeasured at 582 nm.

If a principle of a reaction system is identical, the present method ofthe present invention can be adapted to determination of any substanceother than AMP, for example, guanylate cyclase activity, guanosine3′,5′-cyclic monophosphate (cGMP) and guanosine 3′,5′-monophosphate(GMP) by applying a suitable enzyme corresponding to GMP. The reactionschemes used are shown below.

Step (i)—Cleaning Reaction

As described above, a cleaning mixture of alkaline phosphatase,nucleoside phosphorylase and guanase is used in the Cleaning Reactions.Alkaline phosphatase may be used to dephosphorylate compounds such asglucose-6-phosphate and non-cyclic nucleotides which may increase blankvalues during the measurement of cGMP (Cleaning Reactions).

In the Cleaning Reactions, GTP in a sample is converted to GMP plus 2Pifollowed by conversion to guanosine and Pi. Guanosine is converted intoguanine and ribose-1-posphate and guanine is converted into xanthine andammonia. Likewise in the Cleaning Reaction for determination of cAMP,the Formation of ammonium (or NH⁺⁺) in the final Cleaning Reactionsdrives the series of linked reactions essentially to completion, ensuresremoval of the interfering nucleotides.

Preferably, the enzymes used in the Cleaning Reaction are deactivated,e.g., by heating, prior to the next Step (ii)—Converting Reaction.

Step (ii)—Converting Reaction

The cGMP present in the sample is then converted to GMP withphosphodiesterase. The GMP is combined with ATP in the presence ofguanine monophosphate kinase to yield guanosine-5′-diphosphate (GDP) andADP.

Step (iii)—Cycling Reactions

The GDP is employed in the presence of excess PEP, succinyl-CoA andinorganic phosphate (Pi) to yield an amount of pyruvate (CyclingReactions).

Step (iv)—Detecting Reactions

This pyruvate is quantitated indirectly by adding a known amount of NADHwhich in the presence of acid is converted to lactate and NAD⁺. Thus,the fluorescence of the indicator samples is decreased in directproportion to the amount of pyruvate generated in the sample to beassayed for cGMP, GMP or guanylate cyclase. Also, in order to increasethe assay sensitivity, after decomposition of excess NADH with acid,NAD⁺ formed may be converted with alkali to 2-hydroxy-nicotinic aldehydehaving higher fluorescence intensity. K. Scya et al., Anal. Biochem.,272, 243 (1999).

According to the present invention, an alternative way to measure GMP isto measure an amount of ATP which is decomposed by guanine monophosphatekinase in Step (ii)—Converting Reaction. The amount of ATP can bedetermined by a known method as described above.

In addition to improving assay sensitivity, measurement of adenylatecyclase activity or guanylate cyclase activity with the presentenzymatic assays is significantly less costly, less time consuming,moreover, safer for the operator and better for environment because ofusing no radioactive substance.

Finally, the present assay can readily be adapted to determine theamount of endogenous or exogenous phosphodiesterase in a biologicalsample. That is, a single, preselected amount of cAMP is added to asample containing an unknown concentration of phosphodiesterase. Anyinhibitors specific to phosphodiesterases can be added as needed. Astandard curve is generated by adding a single, preselected excessamount of cAMP to different preselsected, known amounts ofphosphadiesterase. After a finite amount of reaction time (5-60 minutes)in which some but not all of the added cAMP is transformed to AMP by thephosphodiesterase, the reaction is stopped. A cAMP standard curve can berun concurrently to verify that all reactions are working adequately. Acleaning reaction is initiated to degrade all non-cyclic adeninenucleotides. The remaining cAMP will be inversely proportional to thenative plus added phosphodiesterase. Then cAMP is converted to AMP by aconventional method to determine.

Specifically, a tissue sample is dissolved in a SET buffer (Sucrose 0.5M, Tris 0.03 M, EDTA 2 mM) and to the resultant mixture, the doublevolumes of PDE mixture (Tris 50 mM, cAMP 50 AM) is added and incubatedat 37° C. for 20 minutes. The reaction mixture is heated at 80° C. for30 minutes. Cleaning Reaction Mixture in a volume four times as much asa tissue sample is added to the reaction mixture and incubated at 37° C.for 1 hour. The reaction mixture is heated at 80° C. for 30 minutes.Converting Reaction Mixture in a volume ten times as much as a tissuesample is added to the reaction mixture and incubated for 1 hour at roomtemperature. The fluorescence intensity of resultant NADPH is measuredusing Detecting Reactions.

The present invention provides a preparation comprising enzymes orbuffers to be used in a form of kit. The reaction materials of each stepcan be conveniently prepared in a kit, for example, such as CleaningReaction Mixture kit, Converting Reaction Mixture kit, Cycling ReactionMixture kit, Detecting Reaction Mixture kit.

The enzymes used in the present invention are provided in the form of,for example, aqueous solutions, lyophilized or solid formulations in acontainer such as a vial, an ample made by suitable glass or plastic.

The enzymes can be dissolved in buffers, electrolyte, distilled water,or they can be separately put in different containers and can be mixedwith just before use.

Additives such as preservatives, stabilizers, dye stuffs, excipients, pHregulators and the like can be suitably added.

In addition to the amplification reaction of the present invention, theassay as described in the present invention can employ appropriateautomatic analyzers.

The present invention provides a novel, non-radioactive enzymaticfluorometric assay for adenylate cyclase activity and cAMP, as well ascAMP specific phosphodiesterase activity and the biological activity ofthe G regulatory proteins. And also it provides a non-radioactiveenzymatic fluorometric assay for guanylate cyclase activity and cGMP.

The method of the present invention offers several advantages overcurrently available methods compared to conventional methods formeasurement of adenylate cyclase activity and the like. Unlike theassays previously disclosed by Y. Salomon et al., in Anal. Biochem., 58,541 (1974) and Adv. Cyclic Nucleotide Res., 10, 35 (1979), the presentassay does not utilize any radioactive material. In addition, this assayis more sensitive and simpler to perform than the previous assays.

And also, according to the present invention, the reaction period in themethod can be extremely shortened and the operation becomes simplercompared to a method which previously developed by the inventor andothers. See A. Sugiyama et al., Anal. Biochem., 218, 20 (1994); A.Sugiyama et al., J. Clin. Lab., 8, 437 (1994); A. Sugiyama et al., Anal.Biochem., 225, 368 (1995); A. Sugiyama et al., Yamanashi Med. J., 10, 11(1995). In addition, a problem of cloudy in a sample, which is notavoidable in conventional methods, is solved by using a chelating agentsuch as EDTA. And cAMP content in a biological sample in of order of μgcan be correctly measured on a level of pmol or fmol.

The Cleaning Reaction of the present invention can remove all endogenousATP, ADP and AMP which are present in a far higher concentration thancAMP and would otherwise substantially increase the blank. Additionally,the Cleaning Reactions can remove all endogenous glucose-6-phosphate andglycogen as interfering substances in the sample. These CleaningReactions are important for improving assay sensitivity.

It has been found that the reaction period of 1 hour by conventionalmethods could be significantly shortened to about 5-10 minutes bydeletion of 5′-nucleotidase from four enzymes, i.e., apyrase,5′-nucleotidase, alkaline phosphatase and adenosine deaminase which havebeen used in the conventional cleaning reactions.

After Cycling Reactions, since Mg²⁺ is trapped by a chelating agent andexcess of enzymes are deactivated without heating, the sample does notget cloudy.

The sensitivity of this assay can also be increased by reducing thereaction volumes. It can also be increased further by varying theconcentrations of glycogen and inorganic phosphate in the reactionmixture, as reported by Meinrich et al. and E. Helmrich et al.,Biochemistry, 52, 647 (1964). According to the present invention,measurement of adenylate cyclase activity is possible in mammaliantissue biopsy samples comprising as little as 10.0 μg of membraneprotein.

Unlike the radioactive methods, where sensitivity is limited by thespecific activity of [α-³²p] cAMP and the volume size forchromatographic separation and the like, there are no significantbarriers to further increasing the sensitivity of the presentfluorometric method. For example, cAMP has been measured over a broadconcentration range of 1 fmol- 1 mmol.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples wherein the enzymes, substrates and cofactors usedwere obtained from Boehringer Mannheim Co., except for apyrase and5′-nucleotidase which were from Sigma Co., St. Louis, Mo. The [α⁻³²P]ATP, ³H-cAMP, and Aquasol scintilation cocktail were purchased from NewEngland Nuclear. Neutral Chromatographic Alumina WN-3 was obtained fromBio-Rad.

Example 1

Determination of Cleaning Reaction Period

A volume of 3 μL of ATP standard (0, 10, 100, 1000 and 10000 pmol/tube)was added into a 10×57 Pyrex® assay tube. While at room temperature, 25μL of Cleaning Reaction Mixture (100 mM Tris-HCl, pH 8.0; 2 mM MgCl₂; 2U/mL apyrase; 10 U/mL adenosine deaminase; 40 U/mL alkaline phosphatase)was added to each assay tube. The mixture was incubated at 37° C. for 0,5, 10, 15 and 20 minutes. Then Step 2—Converting Reactions (Conversionto AMP), Optional Converting Reactions (Conversion to ATP), Step3—Detecting Cycling Reactions 2 were sequentially carried out and anfluorescence intensity at 340 nm was measured to determine an incubationperiod when ATP have completely disappeared. The results are shown inFIG. 1. From the results, it was found that an interfering substance,i.e., ATP, substantially disappeared after at most 10 minutes ofincubation.

Comparing Example

This test was carried out in order to compare Cleaning Reactions of thepresent invention with that of the cleaning reactions containing5′-nucleotidase.

Preparation of a cleaning reaction mixture containing 5′-nucleotidase

Tris-HCl(pH 8.0), MgCl₂, 5′-nucleotidase, Apyrase, Adenosine deaminaseand Alkaline phosphatase were mixed and to the mixture water was addedup to total 25 μL. The cleaning reaction mixture having finalconcentrations as shown in Table 1 was prepared TABLE 1 A cleaningreaction mixture containing 5′-nucleotidase Compound Final ReactionConcentration Tris-HCl pH 8.0 100 mM MgCl₂ 2 mM 5′-nucleotidase 2.5 U/mLApyrase 2 U/mL Adenosine deaminase 10 U/mL Alkaline phosphatase 20 U/mLWater up to a total volume 25 μL

The cleaning reactions can be shown in the following reaction schemes.

Determination of the Cleaning Reaction Period

Cleaning reactions were carried out using the cleaning reaction mixtureprepared above as described in Example 1.

A volume of 3 μL of ATP standard (0, 10, 100, 1000 and 10000 pmol/tube)was added into a 10×57 Pyrex® assay tube. While at room temperature, 25μL of the cleaning reaction mixture (100 mM Tris-HCl, pH 8.0; 2 mMMgCl₂; 2 U/mL apyrase; 2.5 U/mL 5′-nucleotidase; 0.1 U/mL adenosinedeaminase; 20 U/mL alkaline phosphatase) was added to each assay tube.The mixture was incubated at 37° C. Then Step 2—Converting Reactions,Cycling Reactions and Detecting Reactions as described in Example 2below were carried out and an absorbance at 340 nm was measured todetermine an incubation period when ATP have completely disappeared. Asthe results, it was found that ATP had completely disappeared afterabout 1 hour of incubation.

Example 2

Micro cAMP measurement in Tissue Cultures with Enzymatic Fluorometricassay

(1) Preparation of Samples and Reaction Mixtures

A. Preparation of Biological samples (Ventricular myocyte preparation)

Isolated ventricular myocytes were obtained from hearts of 1 day-oldrats, grown in primary cultures. There were approximately 5 millionviable myocardial cells per heart. After plating at a density ofapproximately 1 million cells per 100 mm, dish cells were grown inminimum essential medium with Hanks balanced salt solution containing 5%bovine calf serum cells. Simpson et al., Cir. Res., 51, 787 (1982). Onday 4, the medium was changed. The cultures contained>90% myocardialcells and cell numbers were constant over time. Simpson et al., Cir.Res., 51, 787 (1982); Rocha-Singh et al., J. Clin. invest., 88, 204(1991); and Rocha-Singh et al., J. Clin. Invest., 88, 706 (1991)

Six plates of myocytes were randomized into two groups (n=3plates/group). The phosphodiesterase inhibitor3-isobutyl-1-methylxanthine (IBMX) was added to the medium of each groupto achieve a 2 μM final concentration. After 5 minutes, isoproterenolwas added to the stimulated group to achieve a final 1 μM concentration,while the control group received no additional drug. After 5 minutes,cells from all six plates and plates of culture medium without cellswere eluted with a total of 5 mL of 100% ethanol. One-tenth of theeluent(0.5 mL) from each dish was removed and air dried in a 12×75 mmborosilicate glass tube and stored at −80° C. The other 4.5 mL weresimilarly air dried and stored. At the time of assay, the pellet wasthawed and resuspended in 100 μL of 0.5 N perchloric acid. The extractswere stirred at 4° C. for 2 minutes and sonicated for 1 minute using asonicator (Branson Cleaning Equipment Co. A Smith-Kline Co., USA). Theextract was neutralized with 25 μL of 2N KOH, centrifuged at 2000 g for30 minutes and 80 μL of supernatant was removed for assay.

B. Preparation of Cleaning Reaction Mixture (See Step 1—CleaningReactions)

One molar Tris-HCl(pH 8.0) 400 μL ; 0.2 mM MgCl₂40 μL; 500 U/mL apyrase32 μL; 400 U/mL adenosine deaminase 100 μL; 4000 U/mL alkalinephosphatase 4 μL were mixed and to the mixture water was added up to atotal volume 4 mL to prepare Cleaning Reaction Mixture (Table 2) TABLE 2Cleaning Reaction Mixture Compound Final Reaction Concentration Tris-HClpH 8.0 100 mM MgCl₂ 2 mM Apyrase 4 U/mL Adenosine deaminase 10 U/mLAlkaline phosphatase 40 U/mL Water up to a total volume 4 mL

After cooling rat ventricular myocyte preparation prepared in Example 2(1) on ice, 0.4 μL of Reaction Mixture of Step 1—Optional CleaningReaction (100 mM Tris-HCl, pH 8.0; 3 mM MgCl₂3 U/mL apyrase; 150 μg/mLadenosine deaminase; 30 U/mL alkaline phosphatase and 75 μg/mL glycogenPhosphorylase-α) was added to the preparation to remove all endogenousadenine nucleotides, glycogen and glucose-6-phosphate. After incubationat 37° C. for 1 hour, enzymes were deactivated by heating at 80° C. for30 minutes.

C. Preparation of Converting Reaction Mixture (See Step 2—ConvertingReactions and Step 2—Optional Converting Reactions)

One molar Tris-HCl(pH 8.0) 400 μL, 0.2 mM MgCl₂40 μL, 4% bovine SerumAlbumin 10 μL, 1 M KCl 600 μL, 0.01 M dithiothreitol 80 μL, 0.1 μM ATP16 μL, 0.5 M Mphosphoenol-pyruvate 24 μL, 2 U/mL Phosphodiesterase 24μL, 720 U/mL Myokinase 24 μL and 2000 U/mL Pyruvate kinase 160 μL weremixed and to the mixture water was added up to a total volume 4 mL toprepare Converting Reaction Mixture (Table 3). TABLE 3 ConvertingReaction Mixture Compound Final Reaction Concentration Tris-HCl pH 8.0100 mM MgCl₂ 2 mM Bovine Serum Albumin 0.01% KCl 150 mM Dithiothreitol 2mM ATP 40 nM Phosphoenolpyruvate 3 mM Phosphodiesterase 12 mU/mLMyokinase 4.5 U/mL Pyruvate kinase 80 U/mL Water up to a total volume 4mL

D. Preparation of Cycling Reaction Mixture (See Step 3—Detective CyclingReactions 2)

One molar Tris-HCl(pH 8.0) 400 μL, 0.2 mM MgCl₂40 μL, 4% bovine SerumAlbumin 10 μL, 0.1 M Fructose 30 μL, 1500 U/mL Hexokinase 160 μL weremixed and to the mixture water was added up to a total volume 4 mL toprepare Cycling Reaction Mixture (Table 4). TABLE 4 Cycling ReactionMixture Compound Final Reaction Concentration Tris-HCl pH 8.0 100 mMMgCl₂ 2 mM Bovine Serum Albumin 0.01% Fructose 3 mM Hexokinase 60 mMWater up to a total volume 4 mL

E. Preparation of Detecting Reaction Mixture (See Step 3—DetectingReaction 2)

One molar Tris-HCl(pH 8.0) 2000 μL, 0.2 mM EDTA 320 μL, 0.1 M NADP⁺ 120μL, 3500 U/mL Phosphoglucose isomerase 6 μL, 1750 U/mLGlucose-6-phosphate dehydrogenase 6 μL were mixed and to the mixturewater was added up to a total volume 4 mL to prepare Cycling ReactionMixture (Table 5). TABLE 5 Detecting Reaction Mixture Compound FinalReaction Concentration Tris-HCl pH 8.0 100 mM EDTA 3.2 mM NADP⁺ 0.01%Phosphoglucose isomerase 1 U/mL Glucose-6-phosphate dehydrogenase 0.5U/mL Water up to a total volume 4 mL

(2) Micro cAMP measurement in Tissue Cultures with EnzymaticFluorometric assay

According to Enzymatic Fluorometric assay comprising a series ofCleaning Reactions, Converting Reactions, Cycling Reactions andDetecting Reactions, cAMP in the ventricular myocyte preparationprepared in (A) as a sample was measured.]

1) Cleaning Reactions (Step 1—Cleaning Reactions)

A volume of 3 μL of neutralized myocyte extract (either 2.4% or 0.24% oftotal eluent from 100 mm plate) or 3 μL of ATP standard (0, 3.6, 7.2,14.4, 21.6 and 28.8 pmol/20 μL) was added into a 10×57 Pyrex® assay tube(Iwaki glass Co.). While at room temperature, 25 μL of Cleaning ReactionMixture prepared from (B) (100 mM Tris-HCl, pH 8.0; 2 mM MgCl₂; 2 U/mLapyrase; 10 U/mL adenosine deaminase; 40 U/mL alkaline phosphatase) wasadded to each assay tube. The mixture was incubated at 37° C. or 10minutes. Enzymes were then deactivated by heating for 30 minutes at 90°C. An internal tissue blank control was similarly prepared.

2) Converting Reactions (Step 2—Converting Reactions (conversion to AMP)and —Optional Converting Reactions (conversion to ATP))

A volume of 25 μL of the Converting Reaction Mixture prepared from (C)(100 mM Tris-HCl, pH 8.0; 2 MM MgCl₂; 0.01% bovine Serum Albumin; 150 mMKCl; 2 mM dithiothreitol; 40 nM ATP; 3 mM phosphoenol-pyruvate; 12 U/mLphosphodiesterase; 4.5 U/mL mvokinase; and 80 U/mL Pyruvate kinase) wasadded to each assay tube from Cleaning Reactions. The mixture wasincubated at room temperature overnight. The reactions were terminatedby heating the assay tubes at 90° for 5 minutes.

3) Cycling Reactions (Step 3—Detecting Cycling Reactions 2)

A volume of 25 μL of the Cycling Reaction Mixture prepared from (D) (100mM Tris-HCl(pH 8.0); 2 mM MgCl₂; 0.01% bovine Serum Albumin; 3 mMFructose; 60 U/mL Hexokinase) was added to each assay tube fromConverting Reactions. In view of the high concentrations of enzymes perreaction, it was important to set up these reactions at 0° C. to insurethe same starting time for all assay tubes. The mixture was incubated at37° C. for 2 hours (about 4000-10000 cycles).

4) Detecting Reactions (Step 3—Detecting Reactions 2)

A volume of 125 μL of the Detecting Reaction Mixture prepared from (E)(100 mM Tris-HCl, pH 8.0; 3.2 mM EDTA; 0.6 mM NADP⁺; 1 U/mLPhosphoglucose isomerase; 0.5 U/mL Glucose-6-phosphate dehydrogenase)was added to each assay tube from Cycling Reactions. After 20 minutes atroom temperature, the final concentration of NADPH was measured using afluorometer (Optical Technology Devises, Inc., NY). The fluorometer wasset such that a reading of 10 fluorometric units was equivalent to 1nmol of NADPH in 900 μL of buffer (50 mM Tris-HCl, pH 8.0).

Individual assay steps were verified by concurrently assaying internalcontrols using known concentrations of the appropriate substrate, forexample, additional ATP was used to check Cleaning Reactions, an AMPstandard curve was used to assess Converting Reactions and an ATPstandard curve was used to assess Cycling Reactions.)

5) Results

Measurement of the absorbance from cAMP standard (0, 3.6, 7.2, 14.4,21.6 and 28.8 pmol/20 μL) at 340 nm afforded the cAMP standard curveshown in FIG. 2.

cAMP values of control group received no additional drug andisoproterenol-stimulated cells from (1) (A) were measured for one-tenthof the eluent from each plate of cells using the cAMP standard curve(FIG. 2) to give an actual value of cAMP 680 pmol/100 mm plate.

The results of measurement of cAMP content using the fluorometric assayof the present invention, immunocolorimetric assay (AmershamInternational Co.) and radioimmunoassay (Amersham International Co.)were shown in FIG. 3. The data generated from measurements of cAMP aresufficiently coincident with the data obtained from a commerciallyavailable radioactive assay with variability usually less than 5%.

In addition, adenylate cyclase activity were measured from the resultsof quantitative measurement of cAMP using the assay of the presentinvention, radioimmunoassay and Salomon method. The data from threemethods were shown in FIG. 4. The values of adenylate cyclase activityare assessed by cAMP amounts (pmol) per minute generated from protein 1mg.

Example 3

Each appropriate amount of 1 M Tris-HCl pH 8.0, 0.2 mM MgCl₂, 1 MK₂HPO₄, 0.1 mM AMP, 4000 U/mL alkaline phosphatase, and 10 mg/mLglycogen phosphorylase were mixed and to the mixture water is added toprepare Cleaning Reaction Mixture in final concentrations as shownbelow. TABLE 6 Cleaning Reaction Mixture Compound Final ReactionConcentration Tris-HCl pH 8.0 100 mM MgCl₂ 2 mM K₂HPO₄ 5 mM AMP 0.1 mMAlkaline phosphatase 20 U/mL Glycogen phosphorylase 10 μg/mL Water —

Each appropriate amount of 1 M Tris-HCl (pH 8.0), 0.1 mM MgCl₂, 1 MK₂HPO₄, 0.1 M AMP, 0.1 M dithiothreitol, 1 mM glucose-1,6-diphosphate,4% bovine Serum Albumin, 0.1 M NADP, and 10 mg/mL Glycogenphosphorylase, 10 mg/mL phosphoglucoMutase, and 5 mg/mLGlucose-6-phosphate dehydrogenase were mixed and to the mixture water isadded to prepare Converting Reaction Mixture in final concentrations asshown below. TABLE 7 Converting Reaction Mixture Compound Final ReactionConcentration Tris-HCl pH 8.0 100 mM MgCl₂ 2 mM K₂HPO₄ 5 mM AMP 0.1 mMDithiothreitol 1 mM Glucose-1,6-diphosphate 4 μM Bovine Serum Albumin0.01% NADP 0.2 mM Glycogen phosphorylase 20 μg/mL Phosphoglucomutase 4μg/mL Glucose-6-phosphate dehydrogenase 2 μg/mL Water —

1) Cleaning Reactions (Step 1—Optional Cleaning Reactions 2)

An aqueous glycogen solution comprising 206 μM of glycogen was preparedand distributed into 12 Pyrex® assay tubes (Iwaki Glass Co., 10×57 mm),each three tubes in an amount of 0, 20, 40 and 80 μL, and water wasadded to each assay tube up to a total volume 80 μL. At roomtemperature, 500 μL of Cleaning Reaction Mixture (100 mM Tris-HCl, pH8.0; 2 mM MgCl₂; 5 mM K₂HPO₄; 0.1 mM AMP; 20 U/mL Alkaline phosphatase;and 10 μg/mL Glycogen phosphorylase) was added to each assay tube. Themixture were incubated at 37° C. for 60 minutes.

2) Detecting Reactions (Step 3—Detecting Reactions 1)

A volume of 500 μL of the converting Reaction Mixture (100 mM Tris-HCl,pH 8.0; 2 mM MgCl₂; 5 mM K₂HPO₄; 0.1 mM AMP; 1 mM dithiothreitol, 4 μMglucose-1,6-diphosphate, 0.01% bovine Serum Albumin, 0.2 mM NADP, and 20μg/mL glycogen phosphorylase, 4 μg/mL phosphoglucomutase and 2 μg/mLglucose-6-phosphate dehydrogenase) was added to each assay tube. Themixture was incubated at 37° C. for 30 minutes. The fluorescencewavelength of 460 nm for each assay tube was measured at a exitingwavelength of 340 nm. In result, the data confirmed that glycogen in theglycogen solution was completely removed by the Cleaning Reactions.

Example 4

Preventive Effect Against Clouding by Chelate Agents

A volume of 125 μL of Detecting Reaction Mixture without EDTA (100 mMTris-HCl, pH 8.0; 0.6 M NADP⁺; 1 U/mL Phosphoglucose isomerase, 0.5 U/mLGlucose-6-phosphate dehydrogenase) was added to the assay tube afterCycling Reactions in Example 2. The mixture was incubated at 90° C. for30 minutes and the enzymes were deactivated. Clouding in the assaysolution was surveyed with eyes. The solution from Example 2 to whichwas added Detecting Mixture with EDTA was employed as a control. Theenzymes in the control were deactivated by EDTA at room temperature.

In result, while the control was clear, the mixture deactivated byheating got cloudy.

INDUSTRIAL APPLICABILITY

The present invention provides a method of determining cAMP content oran adenylate cyclase activity in a biological sample without the use ofradioactive agents.

That is, the cleaning reactions according to the present invention canremove selectively and efficiently endogenous non-cyclic adeninenucleotides such as ATP, ADP and AMP, and glucose-6-phosphate, which actas interfering substances in assay of cAMP. After conversion of cAMP ina sample into AMP, AMP is enzymatically converted into ATP.

ATP is then converted via fructose-6-phosphate into glucose-6-phosphatein order to obtain NADPH, the concentration of NADPH is finallydetermined by a fluorometric method. By the correlation with a finalconcentration of NADPH, a cAMP content or adenylate cyclase activity canbe safely obtained only by enzymatic or chemical reactions without theuse of radioactive agents.

In particular, according to the present invention, the enzymes used inthe cleaning reactions are limited to apyrase, alkaline phosphatase andadenosine phosphatase. Also, by adjustment of their concentrations, thepresent invention succeeds in making the processes for determinationbecome much simpler and in making their reaction periods be extremelyshortened within about 5 to 10 minutes, i.e., by from a twelfth to asixth compared to conventional methods.

Since the cAMP content depends on eutrophy, proliferation,differentiation, adaptation of cells and changes in sensitivity,measurement of cAMP provides a useful way to assess cell viability,endocrine hormonal axis function, adenylate cyclase activity andphosphodiesterase activity. Therefore, the value of cAMP content can bean excellent index in the fields of basic and clinical medicine.

According to the method of the present Invention, cAMP content andadenylate cyclase activity corresponding to cAMP can be determinedsafely and in high sensitivity.

1-20. (canceled)
 21. A kit for determining cAMP content or an adenylatecyclase activity in a biological sample which comprises: (1) a vial forCleaning Reaction comprising effective amounts of apyrase, alkalinephosphatase and adenosine deaminase to remove non-cyclic adeninenucleotides consisting of endogenous ATP, ADP and AMP, and endogenousglucose-6-phosphate in a biological sample; (2) a vial for ConvertingReaction comprising an effective amount of phosphodiesterase toenzymatically convert cAMP in a biological sample into AMP; and (3) avial for Detecting Reaction comprising glycogen, inorganic phosphoricacid and glycogen phosphorylase to convert glycogen intoglucose-l-phosphoric acid; phosphoglucomutase to convertglucose-l-phosphate into glucose-6-phosphate; and glucose-6-phosphatedehydrogenase and NADP⁺ to convert glucose-6-phosphate into6-phosphogluconolactone and NADPH.
 22. A kit for determining cAMPcontent or adenylate cyclase activity in a biological sample whichcomprises: (1) a vial for Cleaning Reaction comprising effective amountsof apyrase, alkaline phosphatase and adenosine deaminase toenzymatically remove endogenous non-cyclic adenine nucleotides otherthan cAMP, and endogenous glucose-6-phosphate in a biological sample;(2) a vial for Converting Reaction comprising effective amounts ofphosphodiesterase, ATP, myokinase, phosphoenolpyruvic acid and pyruvatekinase to enzymatically convert cAMP in the biological sample into ATP;and (3) a vial for Detecting Reaction comprising fructose and hexokinaseto convert ATP into fructose-6-phosphate; phosphoglucose isomerase.glucose-6-phosphate dehydrogenase and NADPT⁺ to convertfructose-6-phosphate into 6-phosphogluconolactone and NADPH.